Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

Simon Duval, Karamatullah Danyal, Sudipta Shaw, Anna K. Lytle, Dennis R. Dean, Brian M. Hoffman, Edwin Antony, Lance C. Seefeldt

Research output: Contribution to journalArticlepeer-review

53 Scopus citations

Abstract

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured firstorder rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s?1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s?1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s?1, 25 °C), (iii) Phosphate release (kPi = 16 s?1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s?1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein-protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein.

Original languageEnglish (US)
Pages (from-to)16414-16419
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number41
DOIs
StatePublished - Oct 8 2013

Keywords

  • Metalloprotein
  • Nitrogen fixation

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